The present invention relates to the genetic engineering of plants in general and relates, in particular, to the method for transforming orchid species using particle-mediated transformation techniques.
There is substantial interest in genetic improvement of orchid species. Orchids are commercially grown globally and are the largest family of flowering plants with more than 800 genera and over 25,000 species. The potential for genetic modification of orchids for disease- and stress-resistance, inducing precocious flowering and developing varieties with modified flower color and morphology is of commercial importance. Conventional plant breeding methods for orchid improvement have been limited mainly due to the prohibitively long reproduction cycles (several years), slow seed maturation (several months), and difficulty of seedling analysis. Therefore, the possibility of employing genetic engineering techniques with orchids appears as an attractive alternative.
Genetic engineering is a means for inserting new genes that confer traits not readily available through conventional breeding. In contrast to sexual crosses, it allows new genes to be added while the genotypes of elite clones are retained intact. In order for genetic engineering approach to be successful, it is imperative to have an efficient gene transfer system for the given species. There are two means for the transformation of plants: use of biological vectors to deliver DNA to cells, primarily via a soil-dwelling plant pathogen bacterium Agrobacterium; and direct delivery of naked DNA into plant cells, primarily via particle bombardment.
The most widely used transformation technique to date is based on the use of the soil-born plant pathogen Agrobacterium, which has the innate ability to transfer a segment of DNA from their large endogenous Ti (tumor-inducing) or Ri (root-inducing) plasmids into an infected plant cell. However, successful application of the techniques depends on the host specific range of Agrobacterium. In the search for species- and genotype-independent transformation methods, several techniques based on the direct delivery of naked DNA to the plant cells were developed. These methods include electroporation, microinjection, PEG- or liposome-mediated DNA uptake, silicon carbide whiskers, and particle bombardment. Particle bombardment technique, also known as biolistics or microprojectiles, is based on the coating of DNA onto small carrier particles, which are then physically accelerated into plant cells. This method has several advantages. First, it does not require removal of cell walls for DNA entry. Second, DNA can be introduced into organized and differentiating cell masses such as meristems and adventitious buds. Third, manipulation of specialized biological plasmids is not required. Finally, DNA transfer does not depend on recognition and binding of a biological vector to cell membranes.
Current orchid propagation and breeding rely heavily on the culture of both seeds and protocorm-like bodies (PLBs) derived from shoot tip meristems. Protocorm, a storage organ, is formed from germinating embryo and possesses an apical meristem and a leaf primodium. PLB, also known as a somatic protocorm, is derived from in vitro culture of apical or axillary bud meristems and resembles the seedling protocorm both functionally and structurally. Such meristematic tissues may be suitable for particle bombardment and recovery of transgenic plants. It has been demonstrated in herbaceous plant species that those bombarded cells can be transformed in a fashion similar to Agrobacterium transformation (Klein et al., Proc. Natl. Acad. Sci. U.S.A., 88, pages 8502-8505, 1988). Germline cells of soybeans has been transformed by this particle-mediated transformation technique (McCabe et al., Bio/Technology, 6, pages 923-926, 1988). The U.S. Pat. No. 5,015,580 describes this technique. The U.S. Pat. No. 5,681,730 also describes the genetic engineering of somatic embryos and plants of gymnosperm species of tree through the use of particle bombardment. The advantage in this approach is that shoots develop directly from the primary and secondary meristems without an intervening explant-organogenesis phase. This minimizes treatment with phytohormones and thus opportunity for somaclonal variation.
Little information is available as to how the particle bombardment-mediated transformation methods might be applied to the transformation of orchids, particularly the members of genus Cymbidium. Currently, only two research papers concerning orchid transformation are available for the members of Dendrobium, none for Cymbidium that holds higher commercial value. Kuehnle and Sugii, Plant Cell Reports, 1992, reported obtaining transgenic Dendrobium. However, they did not show the expression of the screenable marker gene such as GUS. Furthermore, it was not known whether the transgenic plants were chimeric or not. Chia et al., Plant Journal, 1994, transformed Dendrobium orchid using particle bombardment. They developed an alternative selection method that relies on the expression of the introduced firefly luciferase gene, instead of antibiotic selection. In this system, light-emitting cell clumps were screened and isolated using a photon-counting video camera-photomultiplier system and high-power dissecting microscope. The whole screening and isolation process is repeated until a pure transgenic cell line is isolated. The isolated cell line is then induced to regenerate into whole plant. Because of the expensive equipment requirement, time-consuming and microscopic screening of transgenic cells, and the characteristic slow regeneration growth of orchids, this technique seems to be unrealistic for practical use of genetic transformation approach for orchid improvement.
Orchids are substantially different from other plants in their requirements for a transforming system. First, the orchid cells have a low rate of proliferation. Second, the orchid cells are recalcitrant to tissue cultural manipulations. Third, plant regeneration from de-differentiated cells has not been achieved for orchids. Fourth, the orchid cells are less sensitive to antibiotic selection. Orchid is known to be resistant to commonly used aminoglycosides such as kanamycin. It often requires higher than 500 mg/l kanamycin to select transgenic cells (Chia et al., Plant Journal, 1994). Fourth, the orchid cells in tissue culture exude a large quantity of phenolics, oxidized products of which are toxic to the cells. Finally, because of the multicellular structure of meristems, the resulting transgenic plants can sometime be chimeras with transformed and non-transformed sectors. One approach to obtain homogeneously transformed individuals from bombarded meristematic tissues is by selfing the treated generation and selecting for the added trait. The other is to insert DNA into meristematic tissues in early stages of organization and then stimulate continued meristemoid development during antibiotic selection. Application of these approaches to orchid transformation is limited mainly due to the long generation time and slow growth in tissue culture. Therefore, no protocol has been made available for practical use of genetic engineering techniques in orchids, particularly in the genus Cymbidium.
Accordingly, an object of the present invention is to provide an efficient method for transforming orchids, particularly the members of genus Cymbidium with particle bombardment. Another object of the present invention is to provide a method of stimulating continued meristemoid development before and after particle bombardment for transforming the developing protocorm-like bodies. Another object of the present invention is to provide a method of transforming protocorm-like bodies in which the tissues are capable of subsequently undergoing morphogenesis.
Another object of the present invention is to provide a methodology having broad applicability to the genetic engineering of orchids so that orchid improvement can be accelerated.